Proton magnetic resonance imaging is a relatively new diagnostic technique in the field of medical imaging of the body's internal structure. Magnetic resonance (MR) images of the human body are obtained by exposing the protons, that is, hydrogen atom nuclei, contained in the water in tissue to the combined action of high magnetic fields and radio frequency waves. The MR image, derived from the MR signals, depends on the density of the protons in a given tissue, and on the two relaxation parameters of these protons which are referred to as T1 and T2.
In the human body, the most intense T1 signal is obtained from fatty tissue due to low concentrations of water whereas tissues containing high concentrations of water, as for example, cerebrospina fluid and edematous tissue, provide a T1 signal of low intensity. Compartments containing high concentrations of proteins, such as the blood stream and muscle tissue, are associated with an intermediate T1 signal intensity. The administration of a paramagnetic ion into a specific compartment will alter the T1 proton relaxation. The introduction of the magnetic field associated with one or more unpaired electrons will alter the interactions between the protons and their environment. As a result, the T1 relaxation time of the protons will be shortened. The magnitude of this change is dependent on the relative concentration of both protons and the paramagnetic ion.
Paramagnetic ions such as iron, manganese and gadolinium have been utilized as contrast enhancers. Of these ions, gadolinium, by virtue of its seven unpaired electrons, has the largest effect on the T1 value of protons. Accordingly, this ion has been utilized extensively to achieve contrast enhancement. Gadolinium does not occur in the human body and is associated with considerable toxicity when injected into animals as a salt solution, such as gadolinium chloride (Gibby, et al., Investig. Radiol. 25: 164-172 (1990)). To decrease the toxicity of gadolinium, the ion is normally administered in a complex form using organic chelators. Iron and manganese, although naturally occurring in biomolecules, are also intrinsically toxic ions. Their toxicity can also be reduced by chelation. In the case of iron, deferoxamine, has been employed to reduce toxicity (Worah, et al., Investig. Radiol. 23: S281-S285 (1988)).
Gadolinium has been detoxified by complexation with ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentacetic acid (DTPA) (Weinmann, et al., A.J.R. 142: 619-624 (1984)). The Gd-DTPA chelate distributes within the extracellular fluid compartment, does not penetrate the blood-brain-barrier, and is rapidly eliminated by the kidney (Schmiedl, et al., A.J.R. 147:1263-1270 (1986)). Accordingly, Gd-DTPA is useful as a contrast agent for urographic imaging, for detecting abnormal capillary permeability from inflammation and tumors, and for assessment of the integrity of the blood-brain-barrier.
However, the use of Gd-DTPA as a contrast agent has limitations. Gd-DTPA is quickly eliminated from the intravascular compartment, about 50% being cleared from the vascular space into the extravascular fluid compartment on the initial pass through the capillaries (Schmiedl, et al., A.J.R. 147: 1263-1270 (1986)). As such, Gd-DTPA cannot provide selective enhancement of the intravascular space such that blood volume or tissue perfusion, for example, may be assessed.
To overcome such limitations and provide a contrast agent capable of intravascular retention, macromolecular components such as proteins and polysaccharides, as for example, albumin, cellulose and molecular weight dextrans having molecular weights of about greater than 50,000 have been attached covalently to DTPA with subsequent chelation to gadolinium (Brasch, et al., In Contrast and Contrast Agents in Magnetic Resonance Imaging, Special Topic Seminar, P. A. Rinck (ed.), European Workshop on Magnetic Resonance in Medicine, Belgium, pp. 74-93 (1989)). Protein-(Gd-DTPA) conjugates have been prepared with human and bovine serum albumin, immunoglobulin G, and fibrinogen. Such contrast agents have shown predominant intravascular distribution and retention (Schmiedl et al., A.J.R. 147: 1263-1270 (1986)); Paajanen, et al., Magn. Reson. in Med. 13: 18-43 (1990)).
It has been found that attaching Gd-DTPA to a serum albumin molecule improves proton relaxation per mole of Gd.sup.+3 over that observed with Gd-DTPA used alone, due to the slower tumbling rates of the protein molecule. For example, an increase of relaxivity values of about 1.4-to 2.0-fold, of about 3-fold, and of 5- to 10-fold over plain Gd-DTPA have been reported (Paajanen, et al., Magn. Reson. in Med. 13: 18-43 (1990)). By contrast, Gd-DTPA polymeric dextrans are only slightly better than Gd-DTPA in relaxation effectiveness on a per Gd ion basis. With polymeric material, however, fewer moles of gadolinium are required to effectively enhance intravascular structures as compared with free Gd-DTPA. In vivo imaging studies have shown that the Gd-DTPA protein conjugate remains mainly in the vascular space for up to 90 minutes after IV injection in rats (Schmiedl, et al., A.J.R. 147: 1263-1270 (1986)).
The use of albumin-(Gd-DTPA) suffers from several drawbacks. Albumin-(Gd-DTPA) is synthesized by reaction of the cyclic anhydride of DTPA with albumin followed by the addition of an excess of Gd+3 ions. The DTPA groups are covalently linked to the amine moieties of albumin, and the Gd+3 ion is chelated in the DTPA ligand (Schmiedl, et al., A.J.R. 147: 1263-1270 (1986)). However, by using a bifunctional chelating agent such as DTPA anhydride, cross-linking of the albumin moiety is likely to occur. Furthermore, since one of the coordination sites on the chelator is altered by this process, the affinity of the gadolinium ion for the protein bound chelator is reduced.
Another drawback of albumin-(Gd-DTPA) conjugates is that the relatively low coupling efficiency of albumin with DTPA requires the injection of a high quantity of human serum albumin. Concern has been expressed regarding the high potential for immunogenic reactions associated with its modified protein matrix.
Dextrans have been cross-linked with DTPA via a polymerization process to form molecules from small particles of 17,000 MW to large insoluble particles. A typical process for cross-linking dextrans with DTPA utilizes the anhydride of DTPA to achieve ester cross-linking of DTPA to dextran (Gibby, et al., Invest. Radiol. 24: 302-309 (1989)). However, since the DTPA anhydride is a bifunctional cross-linking agent, this polymerization process can prove to be difficult and cumbersome. In addition, that method leads to poorly defined products with broad distribution of molecular weights. Further, the solubility of the resulting compound is much lower than that of the starting dextran component. Further, the replacement of two of the five carboxylic acid groups on DTPA with ester cross-links to the polysaccharide results in a significant decrease in affinity of the DTPA dextran conjugate for the bound metal ion. It has been proposed that hydroxyl groups from the polysaccharides may partially compensate for the loss of negative charge, but no data has been offered in support of this hypothesis. (Gibby, et al., Investig. Radiol. 24: 302-309 (1989)).
Attachment of Gd-DTPA to a protein such as albumin or a polysaccharide moiety represents a means for obtaining a contrast agent that distributes in the vascular compartment without specificity. A current focus in MR imaging is on binding paramagnetics to proteins to provide contrast agents which are tissue- or function-specific. For example, a protein-image contrast conjugate has been prepared by combining antibodies with Gd-DTPA. See for example, Shreve, P. and A. M. Aisen, Magn. Reson. Med. 3:336-340 (1986).
One advantage of using gadolinium as the paramagnetic nucleus is the higher relaxivity as compared to ferric iron. As such, a lower concentration of gadolinium need be administered in order to obtain signal enhancement. However, the loss of gadolinium from DTPA is a known occurrence especially in cases where the DTPA anhydride is utilized for polymer attachment. Further, the association constant, or affinity, of gadolinium to DTPA is relatively low at neutral pH and, more importantly, rapidly decreases when the pH is lowered. This characteristic is a significant problem with in vivo administration, particularly during ischemic insults which lead to acidosis and a localized drop in pH to as much as a full pH unit.
Such instability of chelates presents the threat of in vivo dissociation of the metal complex into the potentially toxic form while within the body. Therefore, it is vital that such contrast agents remain stable to ensure that the paramagnetic ion remains in a sequestered, nontoxic form within the body.
Contrast agents containing ferric iron have been used as an alternative to gadolinium. Like gadolinium, however, ferric iron, must be detoxified for internal administration such as by chelation with deferoxamine (desferrioxamine; DFO). The acute and chronic toxicity of deferoxamine is relatively high, potentially causing hypotension when administered intravenously. Ferrioxamine is a stable complex of ferric iron (Fe.sup.+3) and deferoxamine, having a binding constant of about 10.sup.-30 (Hallaway, et al., Proc. Natl. Acad. Sci. (USA) 86: 10108-10112 (1989)). Ferrioxamine (FO) is excreted primarily in the urine which makes it especially useful as an enhancing agent for the urinary tract. Further, it provides identification of local blood-brain-barrier defects and assessment of renal excretory functions (Wesbey, et al., Physiol. Chem. Phys. and Med. NMR 16: 145-155 (1984); Weinman, et al., A.J.R. 142: 619-624 (1984)). Unlike Gd-DTPA which has a plasma half-life of about 20 minutes, FO clearance is biphasic, with an initial phase of about 128 minutes whereby about one-half of the dose is eliminated, followed by a prolonged elimination phase with a half-life of over 7 hours (Worah, et al., Investig. Radiol. 23: 5281-5285 (1988)).
However, the toxicity of ferrioxamine (FO) is similar to that of deferoxamine (Hallaway, et al., Proc. Nat. Acad. Sci. USA 86: 10108-10112 (1989)). Side effects from fast intravenous injection of either compound may lead to dramatic blood pressure drop. (Niedrach, et al., Investig. Radiol. 23: 687-691 (1988)). Accordingly, ferrioxamine as a paramagnetic contrast agent can be used only in very low concentrations and is limited to the urinary excreting system. Furthermore, the relaxivity of ferrioxamine at 20 MHz and at 37 degrees is 1.4 s.sup.-1 mM.sup.-1, which is a factor of 3 lower than Gd-DTPA. Accordingly, ferrioxamine must be injected at a 2-3 times higher dose than gadolinium-containing chelates to produce the same relaxation effects.
Contrast agents comprising para- or ferromagnetic agents bound to proteins such as immunoglobulins, monoclonal antibodies and blood-pool markers have been suggested for use as tumor-specific MR agents (Paajanen, et al., Magn. Reson. Med. 13: 38-43 (1990)). To achieve a high degree of incorporation of iron, the metal chelator was initially attached to polyamino acids such as polylysine, polyglutamic acids, or other organic polymers such as polyacrylic acid (Shreve, P. and A. M. Aisen, Magn. Reson. Med. 3: 336-340 (1986)). Although this process yielded adducts with a high degree of bound iron ions, the method has several drawbacks. In addition to being relatively cumbersome, the final conjugate products comprise profoundly altered antibodies in a structural sense having a decreased immunoreactivity of between about 60-70%. Therefore, what may be gained in signal appears lost in specificity. Furthermore, polyamino acids do not interact as specifically and with as high affinity as chelators such as deferoxamine. Of particular concern is that loosely bound iron may be associated with considerable toxicity since this form of non-sequestered iron can participate in reactions leading to formation of toxic oxygen- and lipid-derived radicals. Iron bound to deferoxamine, however, cannot participate in such reactions since all coordination sites are occupied.
Therefore, an object of the invention is to provide a macromolecular paramagnetic contrast agent composed of ferric iron for use in magnetic resonance imaging that will enhance proton relaxation times, be free of toxic effects in doses appropriate for contrast enhancement in vivo, remain stable in vivo, retain and/or increase its biological half-life in vivo, and be quickly eliminated from the body after completion of the desired imaging study. Another object is to provide a ferric iron contrast agent which is capable of tissue-specific or compartment-specific distribution in a mammal. Yet another object is to provide a pharmaceutical composition comprising the paramagnetic adduct of the invention and a method of using the composition to enhance magnetic resonance imaging.